Overexpression of many secreted proteins which are stabilized by disulfide bonds cannot be obtained by mere expression in bacterial host cells, due at least to the reducing cytoplasm of E. coli. Such proteins either become degraded or are found insoluble in so-called inclusion bodies. This problem is often addressed by alternative expression strategies such as export of the protein to the periplasm of E. coli or expression in another organism. These strategies are laborious, requiring the recloning of genes of interest in other vectors. In addition, certain proteins of particular interest, e.g., pharmacological interest, cannot currently be produced at high levels an in active form in bacteria.
The following is a summary of the current knowledge in the art regarding the synthesis of disulfide bond containing proteins. The fundamental discovery that a denatured protein, ribonuclease, could assemble correctly in the absence of any catalysts indicated that all the information for the proper folding of a protein was present in its primary amino acid sequence. Since disulfide bonds are necessary for the proper folding of ribonuclease, these experiments were also taken to mean that disulfide bond formation was independent of enzyme catalysts. Thus, it had been presumed that only the presence of oxygen (or small molecules such as oxidized glutathione) is needed in vivo for disulfide bond formation. This presumption appeared to explain the fact that proteins with structural disulfide bonds are only found in the more oxidizing non-cytosolic intracellular compartments or in the extracellular space. According to this view, disulfide bonds do not form in the cytosol simply because the reducing components such as glutathione and thioredoxins keep such bonds reduced.
The first modification of this view of disulfide bond formation and the basis for its compartmentalization came from the finding that disulfide bond formation in gram-negative bacteria does require the presence of a protein catalyst, DsbA (Bardwell, et al. (1991) Cell 67: 581; Kamitani, et al. (1992) EMBO J. 11: 57; Peek, et al. (1992) Proc Natl Acad Sci USA 89: 6210; Tomb, J. F. (1992) Proc Nail Acad Sci USA 89: 10252; Yu, et al. (1992) Mol. Microbiol. 6: 1949). This finding not only changed the picture of how disulfide bond formation takes place normally, but also raised questions about the basis for the absence of disulfide bonds in cytosolic proteins. Normally, the formation of stable disulfide bonds in the cytoplasm is an exceedingly rare event (Locker & Griffiths, (1999) J. Cell Biol. 144: 267). Transient disulfide bonds that are not required for the stability of the native state have been detected in a few cytoplasmic proteins that include enzymes such as ribonucleotide reductase, the transcription factors OxyR and RsrA, the Hsp33 chaperone, and in a partially folded intermediate of the P22 tailspike endorhamnosidase (Aslund, et al. (1999) Proc Nail Acad Sci USA 96: 6161; Robinson & King (1997) Nat. Struct. Biol. 4: 450; Kang, et al. (1999) EMBO J. 18: 4292 and Jakob et al. (1999) Cell 96:341). In general, the oxidation of cysteine thiols in cytoplasmic proteins is strongly disfavored for both thermodynamic and kinetic reasons. First of all, the thiol-disulfide redox potential of the cytoplasm is too low to provide a sufficient driving force for the formation of stable disulfides. Second, under physiological conditions, there are no enzymes that can catalyze protein thiol oxidation. The E. coli cytoplasm contains two thioredoxins, TrxA and TrxC, and three glutaredoxins (Rietsch & Beckwith (1998) Annu. Rev. Genet. 32: 163; Aslund & Beckwith (1999) J. Bacteriol. 181: 1375). The oxidized form of these proteins can catalyze the formation of disulfide bonds in peptides. However, in the cytosol both the thioredoxins and the glutaredoxins are maintained in a reduced state by the action of thioredoxin reductase (TrxB) and glutathione, respectively. In E. coli, glutathione is synthesized by the gshA and gshB gene products. The enzyme glutathione oxidoreductase, the product of the gor gene, is required to reduce oxidized glutathione and complete the catalytic cycle of the glutathione-glutaredoxin system.
In a trxB null mutant, stable disulfide bonds can form in normally secreted proteins, such as alkaline phosphatase, when they are expressed in the cytoplasm without a signal sequence. Subsequent studies revealed that in a trxB mutant, the two thioredoxins are oxidized and serve as catalysts for the formation of disulfide bonds (Stewart, et al. (1998) EMBO J. 17: 5543). Disulfide bond formation was found to be even more efficient in double mutants defective in both the thioredoxin (trxB) and glutathione (gor or gshA) pathways (Prinz, et al. (1997) J. Biol. Chem. 272: 15661). Double mutants, trxB gor or trxB gshA, grow very poorly (doubling time over 300 minutes) and require an exogenous reductant such as dithiothreitol (DTT) to achieve a reasonable growth rate.
In view of the numerous proteins of biotechnological and pharmaceutical interest, that are complex molecules containing multiple disulfide bonds, such as the tissue plasminogen activator (tPA), it would be highly desirable to have an efficient method of production of complicated proteins which retain their biological activity. In addition, since expression of recombinant proteins in bacteria is generally a method of choice, but that the formation of disulfide bonds in recombinant proteins expressed in bacteria has been very inefficient, it would be highly desirable to have a prokaryotic system, e.g., bacterial system that allows efficient expression of recombinant proteins containing multiple disulfide bonds. Such a method would be commercially important, at least in part, to produce therapeutics. For example, tPA, is a widely used therapeutic agent with sales exceeding $400 million per year. However, tPA is currently produced in mammalian cells which are costly to grow, resulting in very high price for the drug (well over $1,000 per dose). Cheaper methods of manufacturing therapeutic proteins would result in increased availability of the drug, to the benefit of many more patients.